The long-term goal of this proposed research is to understand how gene expression is regulated during development. The model system understudy is the Drosophila alcohol dehydrogenase (Adh) gene, a single-copy gene that is transcribed from two promoters into two different ADH mRNAs, the larval and the adult during development. It is proposed that the cis- and trans-acting elements that regulate the developmental expression of Adh be identified and their interactions studied at the molecular level. The cis-acting transcription signals will be identified by comparing the expression of the cloned wild-type Adh to that of specific deletion mutants in the promoter regions, and that of naturally-occurring variant Adh genes cloned from other Drosophila species using an in vivo transient assay in homologous Drosophila cultured cells. The changes in chromatin structure that accompany specific transcription from the larval and the adult promoters will be studied using several nucleases as probes. The transcribing Adh templates will be the transfected exogenous Adh wild-type and specific promoter mutant genes, and the endogenous, in situ, Adh genes of ADH-positive cell lines. Cultured Drosophila cells that express ADH will be analyzed for the presence of factors that bind specifically to the larval and adult promoter regions. Both in vivo assays by transfection and biochemical studies will be used to identify and characterize these putative regulatory factors. Use of the multiple promoters for a single gene is quite common in nature. The knowledge obtained from this study should be of central interest and relevant to other developmental systems.